A general object of the invention is to provide subtilisin mutants which have been mutated such that they do not bind calcium.
Another object of the invention is to provide DNA sequences which upon expression provide for subtilisin mutants which do not bind calcium.
Another object of the invention is to provide subtilisin mutants which comprise specific combinations of mutations which provide for enhanced thermal stability.
Another object of the invention is to provide a method for the synthesis of a subtilisin mutant which does not bind calcium-by the expression of a subtilisin DNA which comprises one or more substitutionxe2x80x99, deletion or addition mutations in a suitable recombinant host cell.
A more specific object of the invention is to provide class I subtilase mutants, in particular BPNxe2x80x2 mutants which have been mutated such that they do not bind calcium.
Another specific object of the invention is to provide DNA sequences which upon expression result in class I subtilase mutants, and in particular BPNxe2x80x2 mutants which do not bind calcium.
Another specific object of the invention is to provide a method for making subtilisin I-S1 or I-S2 mutants, and in particular BPNxe2x80x2 mutants which do not bind calcium by expression of a class I subtilase mutant DNA sequence, and more specifically a BPNxe2x80x2 DNA coding sequence which comprises one or more substitution, addition or deletion mutations in a suitable recombinant host cell.
Yet another specific object of the invention is to provide mutant subtilisin I-S1 or I-S2, and more specifically BPNxe2x80x2 mutants which do not bind calcium and which further comprise particular combinations of mutations which provide for enhanced thermal stability, or which restore cooperativity to the folding reaction.
The subtilisin mutants of the present invention are to be utilized in applications where subtilisins find current usage. Given that these mutants do not bind calcium they should be particularly well suited for use in industrial environments which comprise chelating agents, e.g. detergent compositions, which substantially reduces the activity of wild-type calcium binding subtilisins. 
(1) Field of the Invention
The present invention relates to subtilisin proteins which have been modified to eliminate calcium binding. More particularly, the present invention relates to novel subtilisin I-S1 and I-S2 subtilisin mutants, specifically BPNxe2x80x2 mutants wherein the calcium A-binding loop has been deleted, specifically wherein amino acids 75-83 have been deleted, and which may additionally comprise one or more other mutations, e.g., subtilisin modifications, which provide for enhanced thermal stability and/or mutations which restore cooperativity to the folding reaction.
(2) Description of the Related Art
Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. A well known example thereof, subtilisin BPNxe2x80x2 is a 275 amino acid serine protease secreted by Bacillus amyloliquefaciens. This enzyme is of considerable industrial importance and has been the subject of numerous protein engineering studies (Siezen et al., Protein Engineering 4:719-737 (1991); Bryan, Pharmaceutical Biotechnology 3(B): 147181 (1992); Wells et al., Trends Biochem. Sci. 13:291-297 (1988)). The amino acid sequence for subtilisin BPNxe2x80x2 is known in the art and may be found in Vasantha et al., J. Bacteriol. 159:811-819 (1984). The amino acid sequence as found therein is hereby incorporated by reference [SEQUENCE ID NO:1]. Throughout the application, when Applicants refer to the amino acid sequence of subtilisin BPNxe2x80x2 or its mutants, they are referring to the amino acid sequence as listed therein.
Subtilisin is a serine protease produced by Gram positive bacteria or by fungi. The amino acid sequences of numerous subtilisins are known. (Siezen et al., Protein Engineering 4:719-737 (1991)). These include five subtilisins from Bacillus strains, for example, subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase. (Vasantha et al., xe2x80x9cGene for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open-reading frame between the regions coding for signal sequence and mature protein,xe2x80x9d J. Bacteriol. 159:811-819 (1984); Jacobs et al., xe2x80x9cCloning sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis, Nucleic Acids Res. 13:8913-8926 (1985); Nedkov et al.,xe2x80x9d Determination of the complete amino acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisin BPNxe2x80x2, Carlsberg and amylosacchariticus, Biol. Chem. Hoope-Seyler 366:421-430 (1985); Kurihara et al., xe2x80x9cSubtilisin amylosacchariticus,xe2x80x9d J. Biol. Chem. 247:5619-5631 (1972); and Svendsen et al., xe2x80x9cComplete amino acid sequence of alkaline mesentericopeptidase,xe2x80x9d FEBS Lett. 196:228-232 (1986)).
The amino acid sequences of subtilisins from two fungal proteases are known: proteinase K from Tritirachium albam (Jany et al., xe2x80x9cProteinase K from Tritirachium albam Limber,xe2x80x9d Biol. Chem. Hoppe-Seyler 366:485-492 (1985)) and thermomycolase from the thermophilic fungus, Malbranchea pulchella (Gaucher et al., xe2x80x9cEndopeptidases: Thermomycolin,xe2x80x9d Methods Enzymol. 45:415-433 (1976)).
These enzymes have been shown to be related to subtilisin BPNxe2x80x2, not only through their primary sequences and enzymological properties, but also by comparison of x-ray crystallographic data. (McPhalen et al., xe2x80x9cCrystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg,xe2x80x9d FEBS Lett., 188:55-58 (1985) and Pahler et al., xe2x80x9cThree-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin,xe2x80x9d EMBO J. 3:1311-1314 (1984)).
Subtilisin BPNxe2x80x2 is an example of a particular subtilisin gene secreted by Bacillus amyloliquefaciens. This gene has been cloned, sequenced and expressed at high levels from its natural promoter sequences in Bacillus subtilis. The subtilisin BPNxe2x80x2 structure has been highly refined (R=0.14) to 1.3 xc3x85 resolution and has revealed structural details for two ion binding sites (Finzel et al., J. Cell. Biochem. Suppl. 10A:272 (1986); Pantoliano et al., Biochemistry 27:8311-8317 (1988); McPhalen et al., Biochemistry 27: 6582-6598 (1988)). One of these (site A) binds Ca2+ with high affinity and is located near the N-terminus, while the other (site B) binds calcium and other cations much more weakly and is located about 32 A away (FIG. 1). Structural evidence for two calcium binding sites was also reported by Bode et al., Eur. J. Biochem. 166:673-692 (1987) for the homologous enzyme, subtilisin Carlsberg.
Further in this regard, the primary calcium binding site in all of the subtilisins in groups I-S1 and I-S2 (Siezen et al., 1991, Table 7) are formed from almost identical nine residue loops in the identical position of helix C. X-ray structures of the I-S1 subtilisins BPNxe2x80x2 and Carlsberg, as well as the I-S2 subtilisin Savinase, have been determined to high resolution. A comparison of these structures demonstrates that all three have almost identical calcium A-sites.
The x-ray structure of the class I subtilase, thermitase from Thermoactinomyces vulgaris, is also known. Though the overall homology of BPNxe2x80x2 to thermitase is much lower than the homology of BPNxe2x80x2 to I-S1 and I-S2 subtilisins, thermitase has been shown to have an analogous calcium A-site. In the case of thermitase, the loop is a ten residue-interruption at the identical site in helix C.
Calcium binding sites are common features of extracellular microbial proteases probably because of their large contribution to both thermodynamic and kinetic stability (Matthews et al., J. Biol. Chem. 249:8030-8044 (1974); Voordouw et al., Biochemistry 15:3716-3724 (1976); Betzel et al., Protein Engineering 3:161-172 (1990); Gros et al., J. Biol. Chem. 266:2953-2961 (1991)). The thermodynamic and kinetic stability of subtilisin is believed to be necessitated by the rigors of the extracellular environment into which subtilisin is secreted, which by virtue of its own presence is protease-filled. Accordingly, high activation barriers to unfolding may be essential to lock the native conformation and prevent transient unfolding and proteolysis.
Unfortunately, the major industrial uses of subtilisins are in environments containing high concentrations of metal chelators, which strip calcium from subtilisin and compromise its stability. It would, therefore, be of great practical significance to create a highly stable subtilisin which is independent of calcium.
The present inventors have previously used several strategies to increase the stability of subtilisin to thermal denaturation by assuming simple thermodynamic models to approximate the unfolding transition (Pantoliano et al., Biochemistry 26:2077-2082 (1987); Pantoliano et al., Biochemistry 27:8311-8317 (1988); Pantoliano et al., Biochemistry 28:7205-7213 (1989); Rollence et al., CRC Crit. Rev. Biotechnol. 8:217-224 (1988). However, improved subtilisin mutants which are stable in industrial environments, e.g., which comprise metal chelators, and which do not bind calcium, are currently not available.
Accordingly, it is an object of the invention to provide mutated or modified subtilisin enzymes, e.g., class I subtilases, which have been modified to eliminate calcium binding. As used in this invention, the term xe2x80x9cmutated or modified subtilisinxe2x80x9d is meant to include any serine protease enzyme which has been modified to eliminate calcium binding. This includes, in particular, subtilisin BPNxe2x80x2 and serine proteases which are homologous to subtilisin BPNxe2x80x2, in particular class I subtilases. However, as used herein, and under the definition of mutated or modified subtilisin enzyme, the mutations of this invention may be introduced into any serine protease which has at least 50%, and preferably 80% amino acid sequence identity with the sequences referenced above for subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, or Savinase and, therefore, may be considered homologous.
The mutated subtilisin enzymes of this invention are more stable in the presence of metal chelators and may also comprise enhanced thermal stability in comparison to native or wild-type subtilisin. Thermal stability is a good indicator of the overall robustness of a protein. Proteins of high thermal stability often are stable in the presence of chaotropic agents, detergents, and under other conditions, which normally tend to inactivate proteins. Thermally stable proteins are, therefore, expected to be useful for many industrial and therapeutic applications in which resistance to high temperature, harsh solvent conditions or extended shelf-life is required.
It has been further discovered that combining individual stabilizing mutations in subtilisin frequently results in approximately additive increases in the free energy of stabilization. Thermodynamic stability has also been shown to be related to resistance to irreversible inactivation at high temperature and high pH. The single-site changes of this invention individually do not exceed a 1.5 Kcal/mol contribution to the free energy of folding. However, these small incremental increases in the free energy of stabilization result in dramatic increases in overall stability when mutations are combined, since the total free energy of folding for most proteins is in the range of 5-15 Kcals/mol (Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman and Company, New York (1984)).
X-ray crystallographic analysis of several combination mutants reveals that conformational changes associated with each mutation tend to be highly localized with minimal distortion of the backbone structure. Thus, very large increases in stability can be achieved with no radical changes in the tertiary protein structure and only minor independent alterations in the amino acid sequence. As previously suggested (Holmes et al, J. Mol. Biol. 160:623 (1982)), contributions to the free energy of stabilization can be gained in different ways, including improved hydrogen bonding and hydrophobic interactions in the folded form and decreased chain entropy of the unfolded enzyme. This is significant since thermostable enzymes generally have more extended half-lives at broader temperature ranaes, thereby improving bio-reactor and shelf-life performance.
As noted supra, the invention provides subtilisin mutants which comprise one or more deletion, substitution or addition mutations which provide for the elimination of calcium binding. Preferably, this will be effected by deletion, substitution or insertion of amino acids into the calcium A-site, which in the case of class I subtilases comprises 9 amino acid residues in helix C. In the case of subtilisin BPNxe2x80x2, the subtilisin mutants will preferably comprise one or more addition, deletion or substitution mutations of the amino acids at positions 75-83, and most preferably will comprise the deletion of amino acids 75-83, of SEQUENCE ID NO: 1. The deletion of amino acids 75-83 has been discovered to effectively eliminate calcium binding to the resultant subtilisin mutant while still providing for subtilisin BPNxe2x80x2 proteins having enzymatic activity.
Such subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID NO: 1 may further include one or more additional amino acid mutations in the sequence, e.g., mutations which provide for reduced proteolysis. It is another object of the invention to produce subtilisin mutants lacking calcium binding activity which have been further mutated to restore cooperativity to the folding reaction and thereby enhance proteolytic stability. It is another object of the invention to provide thermostable subtilisin mutants which further do not bind calcium and comprise specific combinations of mutations which provide for substantially enhanced thermal stability.
In particular, the subtilisin mutants of the present invention will include subtilisins from Bacillus strains, such as subtilisin BPNxe2x80x2, subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus and subtilisin mesenticopeptidase, which comprise one or more deletion, substitution or addition mutations.
The present invention further provides for subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID NO: 1, which have new protein-protein interactions engineered in the regions around the deletion leading to large improvements in stability. More specifically, mutations at ten specific sites in subtilisin BPNxe2x80x2 and its homologues are provided, seven of which individually, and in combination, have been found to increase the stability of the subtilisin protein. Improved calcium-free subtilisins are thus provided by the present invention.